Variants Of Undetermined Significance Research Articles

Assessment of the Frequency of Variants of Undetermined Significance (VUS) in Adult Patients with Chronic Idiopathic Neutropenia Studied for Myeloid Gene Mutations By Next Generation Sequencing

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnosis of CIN is based on exclusion criteria, namely the absence of clinical/laboratory evidence of any underlying disease or use of drugs that might be associated with neutropenia, absence of history of exposure to irradiation or use of chemical compounds, negative antineutrophil antibodies, and normal bone marrow (BM) morphology and karyotype. We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients and we found that 11.35% of the patients display clonal hematopoiesis in term of presence of pathogenic/likely pathogenic variants with VAF higher than 2%. Variants of undetermined significance (VUS) were ruled out from the analysis and their potential significance was not assessed. Aims: In the current study we have assessed the presence, frequency and potential clinical significance of VUS detected in our cohort of CIN patients previously studied for clonal hematopoiesis. Methods: Genomic DNA was extracted from CIN patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies (Tsaknakis G et al. Blood. 2021;138:1249-1257). Results: NGS analysis of myeloid genes has been performed in 139 CIN patients (106 female subjects and 33 male subjects, aged 21 to 89 years with a median age of 59 years). Re-analysis of patients' NGS data and assessment of VUS revealed that 35 out of 139 patients (25.18%) carried one or more VUS (a total of 39 VUS were detected, all with an allelic frequency of 50% and all missense variants). Genetic variants were evaluated according to the ACMG guidelines and classified as VUS. The majority of VUSs were observed in ATM (n=7) and SETBP1 (n=6) genes followed by TET2, NOTCH1 (4 occurrences respectively) and PLCG2 (n=3). Two different patients shared the same variant in TET2 gene whereas two siblings shared the same variant in SETBP1 gene. Pathogenicity score calculation based on the combined evidence from multiple other in-silico predictors identified 3 variants in 3 different genes as having the highest probability to be pathogenic. Namely these were PLCG2K456E,CXCR4R235C and SF3B1T56I. PLCG2 variants have been implicated in familial cold autoinflammatory syndrome 3 (PLCG2 associated antibody deficiency and immune dysregulation, PLAID) and in autoinflammation, antibody deficiency, and immune dysregulation syndrome (APLAID) whereas CXCR4 variants are known to be associated with Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. SF3B1 mutations have not yet been shown to be involved in hereditary disease. Conclusions: Our study of VUS assessment in a cohort of 139 well characterized adult CIN patients identified 39 VUS, all heterozygous, in a total of 35 patients. Further steps include paired analysis with constitutional reference tissue to determine the germline nature of the mutations. Moreover, these variants should be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to disease onset. This study of VUS analysis in CIN patients is anticipated to contribute to the better understanding of the pathogenetic causes of this rare disease.

ODP208 Hypoglycemia as a Presenting Feature of McArdle's Disease

Abstract Introduction McArdle's disease (glycogen storage disease type V) is an autosomal recessive disease caused by mutations in the gene encoding for the muscle isoform of glycogen phosphorylase (PYGM). Characteristic symptoms include exercise intolerance, myalgia, muscle stiffness. Approximately half develop rhabdomyolysis, myoglobinuria, and renal failure. Hypoglycemia is an uncommon presenting symptom. Case report A 36-year-old male, previous college football athlete presented with jitteriness and blurry vision while exercising over the last one year. Symptoms occur 12-15 minutes into aerobic exercise. Fingerstick blood sugar during these episodes were as low as 30 mg/dl and afterwards the patient reported fatigue all day long. Consuming high carbohydrate foods and fluids supplemented with dextrose prior to exercise mitigate his symptoms. He reported no fasting hypoglycemia. Retrospectively, the patient reports that for years he noticed feeling fatigued a few minutes into starting exerice which improves after short periods of rest "second wind phenomenon." He can perform heavy resistance exercise like weightlifting but struggles with sustained aerobic activity. Past medical history is significant for HLA B27 positivity, alopecia areata and uveitis. Medications include multivitamin and creatine supplements. Physical exam revealed a healthy, well developed muscular individual with no other remarkable findings. Family history is significant for McArdle's disease in his brother, diagnosed at age 20 based on a muscle biopsy after multiple episodes of rhabdomyolysis. Grandfather had diabetes mellitus and pancreatic carcinoma. Labs revealed a creatine kinase (CK) level of 1638.7 U/L (49-397 U/L), CK-MB 49.1 ((0.1-5.9 ng/ml), 8 AM morning cortisol of 12.3 ug/dl (4.2-22.4 ug/dl), A1c 5.3%. There was no myoglobinuria. Thyroid function tests were within normal limits. Investigations done during an episode of exercise induced hypoglycemia - fingerstick blood sugar 44 mg/dl, plasma blood glucose 56 mg/dl(65-99 mg/dl), Insulin <0.5 mU/L(3-25mU/l), C-Peptide 0.5ng/ml(1.1-4.4ng/ml), Proinsulin 1.1 pmol/L(0-10. 0 pmol/L). Insulin autoantibody was negative. This ruled out exercise induced hyperinsulinemia. IGF-1 and IGF-2 levels were normal. Genetic study performed by the Glycogen Storage Disease Panel showed PYGM c.2262del (p. Lys754Asnfs*49), Pathogenic, PYGM c.1537A>G (p. Ile513Val), Uncertain Significance, PYGM c.833G>C (p. Arg278Pro), Uncertain Significance. Subsequently, the patient's brother underwent genetic testing, which confirmed the presence of all three before-mentioned PYGM variants. His father was found to carry the two variants of undetermined significance, suggesting that the patient and his brother inherited the pathogenic variant from their deceased mother. Discussion The patient's medical history, along with his personal and family's genetic findings were found to be consistent with McArdle's disease. Our patient's presentation is unique in that his exercise tolerance is much higher as compared to most individuals with McArdle's and his presenting complaint was hypoglycemia. Additionally, one of the variants of uncertain significance is likely to be recognized as pathogenic in the future. Presentation: No date and time listed

Effects of anxiety and depression on the quality of life of male cancer patients undergoing genetic investigation for hereditary cancer predisposition syndromes.

e22529 Background: Genetic tests have been increasingly requested to identify families at risk of hereditary cancer. In a family with a known mutation, the offspring of a carrier, whether male or female, have a 50% risk of inheriting the mutation. Most studies on the psychological implications associated with genetic testing have focused on women and few studies have focused on the male population. Methods: A total of 56 men with a personal history of cancer underwent screening for mutations in a panel of 31 genes associated with hereditary cancer. Assessment of quality of life (QoL) and levels of anxiety and depression was performed after patients received the genetic test result. For this, the HADS and WHOQOL-BREF scales were used. Wilcoxon, X², and Spearman correlation tests were used (SPSS v20.0 for Windows). Results: Of the 56 participants, 28.6% had a pathogenic mutation, 32.1% had a Variant of Undetermined Significance (VUS), and 39.3% had no mutation. The presence of the mutation was not associated with QoL (p = 0.967) or anxiety (p = 0.436) or depression (p = 0.945). Mean QoL was 77.95±7.38 (range = 50.00-95.20), mean anxiety was 5.14±2.62 (range = 1-14) and mean depression scores were 4.14±2.55 (range = 0-14). QoL was inversely correlated with levels of anxiety (p < 0.001, r = -0.496) and depression (p < 0.001, r = -0.494), parameters that were directly correlated (p = 0.037, r = 0.279). Most patients had QoL > 80 (n = 38, 67.9%), and only 16 (28.6%) and 13 (23.2%) men had HAD scores > 5. Conclusions: The result of carrying a mutation did not influence the quality of life and anxiety or depression levels in the male population evaluated. However, it was observed that levels of anxiety and depression can compromise the quality of life. The data from the present study reinforce the importance of considering the psychological aspects in the process of genetic investigation for hereditary cancer. In addition, it suggests that educational levels must be investigated to assess whether the low levels of anxiety and depression observed are associated with a low level of understanding of the clinical significance of carrying a pathogenic mutation.

252 Whole exome sequencing in epilepsy: quality of reporting in literature

PurposeWhole exome sequencing is a new rapid and cost effective tool for the diagnosis of the epilepsies.MethodOur primary aim was to assess the quality of reporting of WES. Secondary aims; to compare the quality of studies based on journal type if recent ACMG guideless have made influenced reporting quality; if any outcome reporting biases exist.We analysed studies of whole exome sequencing in epilepsy. We used a self-constructed benchmark to quantitatively analyse studies, explore retrospectively if reporting trends have changed.ResultsTotal of 307 studies was included. Reporting of data was heterogenous with poor reporting of diagnostic yield in 6% of studies, incidental findings in 11%, variants of undetermined significance in 19%. Predictors of poor reporting included: Journal type, proband status and case reports. Pairwise compari- sons of genetics journals versus other journals using relative risks yielded significant differences in reporting of consanguinity status (RR 1.27 CI 1.04–1.55); variant filtering (RR 1.95 CI 1.41–2.7) and determination of pathogenicity (RR 1.35 CI 1.17–1.56).ConclusionReporting of WES data in the medical literature is heterogenous, making comparisons across studies difficult. Formal structured guidelines similar to the CONSORT guidelines are needed to address this issue further.arifshukralla@gmail.com

Analysis of 29 Targeted Genes for Non-Obstructive Azoospermia: The Relationship between Genetic Testing and Testicular Histology.

To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology. Forty-eight patients with unexplained NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Zagreb, Croatia for testicular biopsy. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing (NGS) analysis and gene sequencing. We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Exploring phenotypes in hypertrophic cardiomyopathy with machine learning data integration. A multicentric, multimodality pilot study

Abstract Background Interpreting patient phenotypes is a challenge when screening for hypertrophic cardiomyopathy (HCM). Machine learning (ML) can potentially help with advanced data integration - combining information contained in whole-cardiac cycle echo deformation and velocity profiles with standard clinical variables. The aim is to apply an ML approach to integrate whole cardiac cycle echo data with clinical variables to explore HCM phenotypes. Methods The cohort consisted of 138 participants from two centres: HCM patients (n=91) and relatives (n=47). Echocardiography was performed, whereas magnetic resonance and genetic testing in 48% and 82%, respectively. Whole cardiac cycle echo data (mitral and aortic velocity profiles, and six regional left ventricular (LV) deformation curves) were combined with clinical variables (age, sex, heart rate, e' medial and e' lateral) and used as the ML input. An unsupervised ML algorithm created a representative space where participants were positioned based on integrated data, blinded to disease status. Clustering was used to determine phenogroups and estimate the average characteristics. Data on family history (FHx), genotype, arrhythmias or syncope, implantable cardioverter-defibrillators (ICD), and late gadolinium enhancement (LGE) were used to interpret the phenogroups. As the LA diameter was not available in the dataset, the HCM risk for sudden cardiac death (SCD) was not calculated, however, the Table shows relevant variables to infer clinical risk. Results Clustering divided the participants into 6 phenogroups (P1–6) (Figure). Average echo profiles are shown in the Figure, while the clinical data in the Table. P1/2 was defined by symptomatic patients with a high prevalence of positive genotypes, a positive FHx of SCD, and a burden of comorbidities. Echo findings showed pronounced structural/functional remodeling, and P1 was associated with severe septal hypertrophy and outflow tract obstruction. The high prevalence of ICD devices defined P1/2 as high risk groups. In comparison, patients in P3/4 were younger, with milder LV hypertrophy, but still considerable functional impairment. P3 had a higher burden of FHX and a higher prevalence of pathogenic mutations, whereas P4 a higher incidence of hypertension, high heart rate, mitral inflow fusion and findings of LGE. Finally, P5/6 consisted of younger individuals, predominantly HCM relatives, with a mild phenotype and, thus, low inferred risk. As expected, the majority of patients with the genetic variants of undetermined significance were located in P5. Conclusion ML can help derive clinically interpretable phenotypes in HCM based on the automated integration of whole cardiac cycle deformation and velocity data with conventional clinical parameters. The derived phenogroups correspond with established risk profiles in HCM. An expanded dataset is needed to enable further exploration of the phenotype-genotype relations and to define prognostic value. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This work was supported by the Horizon 2020 European Commission Project H2020-MSCA-ITN

Early-Onset Atrial Fibrillation and the Prevalence of Rare Variants in Cardiomyopathy and Arrhythmia Genes

Early-onset atrial fibrillation (AF) can be the initial manifestation of a more serious underlying inherited cardiomyopathy or arrhythmia syndrome. To examine the results of genetic testing for early-onset AF. This prospective, observational cohort study enrolled participants from an academic medical center who had AF diagnosed before 66 years of age and underwent whole genome sequencing through the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine program. Participants were enrolled from November 23, 1999, to June 2, 2015. Data analysis was performed from October 24, 2020, to March 11, 2021. Rare variants identified in a panel of 145 genes that are included on cardiomyopathy and arrhythmia panels used by commercial clinical genetic testing laboratories. Sequencing data were analyzed using an automated process followed by manual review by a panel of independent, blinded reviewers. The primary outcome was classification of rare variants using American College of Medical Genetics and Genomics criteria: benign, likely benign, variant of undetermined significance, likely pathogenic, or pathogenic. Disease-associated variants were defined as pathogenic/likely pathogenic variants in genes associated with autosomal dominant or X-linked dominant disorders. Among 1293 participants (934 [72.2%] male; median [interquartile range] age at enrollment, 56 [48-61] years; median [interquartile range] age at AF diagnosis, 50 [41-56] years), genetic testing identified 131 participants (10.1%) with a disease-associated variant, 812 (62.8%) with a variant of undetermined significance, 92 (7.1%) as heterozygous carriers for an autosomal recessive disorder, and 258 (20.0%) with no suspicious variant. The likelihood of a disease-associated variant was highest in participants with AF diagnosed before the age of 30 years (20 of 119 [16.8%; 95% CI, 10.0%-23.6%]) and lowest after the age of 60 years (8 of 112 [7.1%; 95% CI, 2.4%-11.9%]). Disease-associated variants were more often associated with inherited cardiomyopathy syndromes compared with inherited arrhythmias. The most common genes were TTN (n = 38), MYH7 (n = 18), MYH6 (n = 10), LMNA (n = 9), and KCNQ1 (n = 8). In this cohort study, genetic testing identified a disease-associated variant in 10% of patients with early-onset AF (the percentage was higher if diagnosed before the age of 30 years and lower if diagnosed after the age of 60 years). Most pathogenic/likely pathogenic variants are in genes associated with cardiomyopathy. These results support the use of genetic testing in early-onset AF.

Outcomes of large panel genetic evaluation of breast cancer patients in a community-based cancer institute

IntroductionThe use of restricted versus expanded panel genetic testing in breast cancer is controversial, with some institutions offering predominantly abbreviated gene panel testing. Our community program has offered larger panel testing for several years. We sought to evaluate the outcomes of large panel genetic testing and understand their impact on patient care. MethodsA retrospective review of our multi-institutional tumor registry was performed from 2015 to 2018 for patients undergoing surgery for breast cancer. Referral to genetic counseling and outcomes of panel testing were examined. Results2237 patients met study criteria. Median age was 63 years (range 22–99). Eight hundred and thirty-eight patients (37.4%) were referred for genetic counseling. Of these patients, 509 (60.7%) had negative results, 108 (12.8%) had deleterious mutations (37 not included in abbreviated panels), and 221 (26.3%) had variants of undetermined significance (VUS). Bilateral mastectomy rates for patients with deleterious mutations were 53.7%, versus 31% for negative and 32.6% for VUS. DiscussionLarge panel testing finds a significant number of actionable mutations. The increased identification of VUS did not result in higher mastectomy rates.

Gene diagnosis and pedigree analysis of two Han ethnicity families with propionic acidemia in Fujian.

Propionic acidemia is associated with pathogenic variants in PCCA or PCCB gene. We investigated the potential pathogenic variants in PCCA or PCCB genes in Fujian Han population.Two probands and their families of Han ethnicity containing two generations were subject to newborn screening using tandem mass spectrometry, followed by diagnosis using urine gas chromatography mass spectrometry. Sanger sequencing was used to identify potential mutations in PCCA and PCCB genes.Compound heterozygous variants were identified in PCCB gene in two siblings of the first family, the youngest girl showed a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 and a heterozygous missense variant c.1301C>T (p.Ala434Val) in exon 13, which were inherited respectively from their parents. The oldest boy is a carrier with a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 which were inherited from his father. In the second family, c.1535G>A homozygous mutations were identified in the baby girl, which were inherited respectively from their parents. In silico analysis, several different types of bioinformatic software were utilized, which predicted that the novel variant c.1381G>C in PCCB gene was damaged. According to ACMG principle, the missense variant c.1381G>C (p.Ala461Pro) in exon 13 was a Variant of Undetermined Significance (VUS).One novel missense variant and two missense variants in PCCB gene were identified in the study. The novel variant of PCCB gene identified VUS was identified for the first time in the Chinese population, which enriched the mutational spectrum of PCCB gene.

Genomic characterization and molecular taxonomy of sarcomatoid variant prostate cancer.

168 Background: Sarcomatoid carcinoma is a rare and aggressive histologic variant pattern of prostate cancer with morphologic features associated with both mesenchymal and epithelial tissue elements. While sarcomatoid cancer can be found concurrently with adenocarcinoma in untreated specimens, it is most often encountered in post-treatment biopsies. Limited data is available to categorize genomic alterations in sarcomatoid prostate cancer or to genetically define any potential relationship between sarcomtaoid and adenocarcinoma patterns from the same patient. Methods: Clinical characteristics, histology, and genomic alterations for 5 cases of sarcomatoid prostate cancer and 2193 cases with adenocarcinoma are presented. All cases were analyzed for genomic alterations against a custom panel via Tempus xT tissue biopsy assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth and whole transcriptome RNA sequencing) for germline and/or somatic mutations. The xT assay detects single nucleotide variants, specific insertion/deletions, amplifications and gene fusions, as well as TMB and MSI status. Results: Patterns of genomic alterations in sarcomatoid cancer are tabulated and compared with adenocarcinoma. Of note, BRCA2 alterations were found in 2 of 5 sarcomatoid cases including 1 example of BRCA2 loss and 1 variant of undetermined significance (p.K2316R). Sequential analysis of samples from the subject with BRCA2 loss demonstrates genomic alterations that are shared across multiple samples despite different histologic patterns and following 14 months of treatment with GnRH antagonist and abiraterone (Table). Conclusions: Sarcomatoid prostate cancer shares many genomic alterations with that found in adenocarcinoma. Genomic characterization of this variant form of prostate cancer may reveal clinically actionable genetic changes. [Table: see text]

Mutations of uncertain significance in heterozygous variants as a possible cause of severe short stature: a case report

BackgroundLinear bone growth is achieved by the division of chondrocytes at the growth plate and is regulated by endocrine and paracrine factors such as growth hormone. Mutations that negatively affect chondrogenesis can be a contributor to short stature. One such mutation can occur in the ACAN gene, causing short stature and advanced bone age. Similarly, mutations in growth hormone receptors (GHR) can lead to Laron syndrome (LS), one of the several disorders that are collectively called growth hormone insensitivity syndrome (GHI). Another example is Floating-Harbor syndrome (FHS), a rare autosomal dominant due to mutations in the SRCAP gene that can also result in short stature.Case presentationWe report the case of a 6-year-old female with concomitant mutations in the three genes mentioned above. The mutations reported here were found on genetic studies and are usually benign, causing a variant of undetermined significance. However, our patient’s phenotype could only be explained by the compounded effects of pathogenic mutations of these genes. Some of the same mutations were also found in the patient’s father and her paternal grandfather. Both also presented with short stature, though not to the same degree as our patient. While these mutations are often reported to be insignificant, they gave rise to severe short stature and a specific phenotype in the patient when presented together. We think that even though the GHI spectrum is inherited through an autosomal recessive pattern, the sum of these heterozygous mutations resulted in severe short stature despite the limited GHI seen in our patient, the father, and the grandfather, through a rare ACAN and SRCAP mutation that, to our knowledge, has not been previously reported as a pathogenic mutation in the literature.ConclusionWe investigated the possible synergistic effects of these variations on exacerbation or masking of the signs and symptoms of GHI with the hope of providing a better understanding of these genes and their function through our rare case.

Role of a Rare Variant in APC Gene Promoter 1B Region in Classic Familial Adenomatous Polyposis

Background: Familial adenomatous polyposis (FAP) is most commonly caused by germline variants in the adenomatous polyposis coli (APC) gene. Although definite pathogenic variants could be detected in the majority of individuals with FAP, there are still numerous variant-negative FAP patients. Method: We utilized a 139-gene next-generation sequencing (NGS) panel and multiplex ligation-dependent probe amplification (MLPA) to detect pathogenic variants in FAP patients and found a variant-negative pedigree. Through whole-exome sequencing (WES), we identified a point variant in the noncoding region in the APC gene. Finally, we used Sanger sequencing to analyze its pedigree cosegregation and a dual-luciferase reporter (DLR) assay to assess its function. Results: With the exception of 2 variants of undetermined significance (VUS), WES showed no pathogenic or likely pathogenic variants. After performing MLPA, the pedigree was still variant-negative. Interestingly, through WES, a point variant c.-190G>A located in the promoter 1B region of the APC gene was identified in 3 affected individuals. Moreover, a variant carrier was found during screening of the family with Sanger sequencing. Through the DLR assay, we further confirmed that the variant c.-190G>A caused significant suppression of downstream transcription of APC. Conclusions: The variant (c.-190G>A) in the APC promoter 1B region is able to cause FAP with a classic phenotype, but this kind of variant in the noncoding region could be missed by conventional genetic testing. Thus, utilizing sequencing technologies covering a larger area can help us to further explore the pathogenesis in variant-negative FAP cases.

Association of LRP1B pathogenic genomic alterations with favorable outcomes with immune checkpoint inhibitors across multiple tumor types.

3007 Background: Low-density lipoprotein receptor-related protein 1b (LRP1b) is a putative tumor suppressor and one of the most frequently altered genes in cancer. Our prior single-center work suggested that LRP1B alterations may enrich for responses to immune checkpoint inhibitors (ICI) in solid tumors including prostate cancer; however, validation of these findings is needed. Methods: We conducted a multicenter, retrospective analysis of patients with LRP1B alterations (on tissue-based next-generation sequencing panels) treated with ICI at Duke, Johns Hopkins (JHU), and University of Michigan (UM). The primary objective was to assess the association between objective response rate (ORR) to ICI and pathogenic LRP1B alterations, defined as deletions or truncating alterations, when compared with LRP1B variants of undetermined significance (VUS), defined as missense mutations not predicted to be pathogenic in COSMIC. Missense changes with a COSCMIC FATHMM score of > 0.8 were categorized separately as likely pathogenic. Summary statistics, ORR, progression free survival (PFS), and overall survival (OS) were calculated. Results: 101 patients (44 Duke, 35 JHU, 22 UM) with LRP1B alterations were treated with ICI. Median age was 61 (range 32-82). The most common tumor types by alteration (pathogenic or likely pathogenic/VUS%) were lung (33/47%), GI (17/13%), prostate (11/7%), sarcoma (2/9%), melanoma (11/0%), and others (26/24%). 93% of patients received single-agent PD-(L)1 inhibition. The ORR for patients with either pathogenic/likely pathogenic alterations, or VUS alterations was 57% and 18%, respectively. After excluding MSI-high or TMB-high ( > 10 mut/Mb) tumors, ORR was 14/25 (56%) and 6/36 (17%), respectively. Pathogenic or likely pathogenic LRP1B alterations were associated with longer PFS (HR 0.39, 95% CI 0.24-0.63) and OS (HR 0.58, 95% CI 0.36-0.95). Conclusions: This multicenter study shows impressive and durable objective response rates to ICI for patients harboring pathogenic LRP1B alterations when compared to those with LRP1B VUS, independent of TMB/MSI status. Further mechanistic insights and prospective validation studies are warranted. [Table: see text]

Germline genetic mutational landscape and characteristics of men with prostate cancer (PCa): A single institution experience.

e13679 Background: The role of germline genetic testing (GGT) is rapidly changing for men with PCa. We describe the characteristics of men with PCa diagnosed with pathogenic (P) and likely pathogenic (LP) mutations on GGT. Methods: We conducted a retrospective single-institution study for all men with PCa who received single gene or multi-gene GGT from 1997-2019, unselected for disease stage, Gleason score, or age at diagnosis, and while most had a family history of cancer, some did not. All participants were from the Risk Assessment Program (RAP) database at FCCC and consented for research purposes at the time of GGT. Results: 160 men with PCa had GGT. 72/160 (45%) patients had no mutations. 41/160 (26%) patients had a variant of undetermined significance (VUS) (13 with 2 VUS, 1 with 3 VUS). 47/160 (29%) men were found to have a P or LP germline mutation. 26/47 (55%) with recurrent disease, and 1/47 (2%) unknown. 46 were Caucasian, and 1 was African American ( PALB2). 28/47 (60%) had Gleason Scores (GS) ≤7. 6/47 (13%) had a Prostate Specific Antigen (PSA) at diagnosis < 4.0 ng/mL. 23/47 (49%) were diagnosed < 60 years of age, and 8/47 (17%) at ≤ 50. 19/23 (83%) PCa cases with P/LP diagnosed below age 60 had DNA repair genes: BRCA2 (8), BRCA1 (4), CHEK2 (4), ATM (2), and PALB2 (1). 74/160 (46%) were tested after PCa NCCN genetics guidelines changed (October 2017), of which 19/74 (25%) had P/LP, and of those positive for P/LP, 6/19 (32%) had non-metastatic disease. Two patients had small cell neuroendocrine PCa ( CHEK2 and BRCA2), the remaining had adenocarcinoma PCa. Please see Table for full results. Conclusions: To our knowledge, this is the largest single-institution study reporting PCa characteristics and incidence of P/LP germline mutations. Rates of DNA-repair gene mutations were higher than previously reported, possibly reflecting our specialized cancer center’s population. While BRCA1/2 mutations were the most represented (55%), we describe PCa patients with mutations in ATM, CHEK2, NBN, PALB2, CFTR, and MUTYH, recognizing a few are not known to be linked to PCa. More than half of P/LP mutation carriers had GS ≤7. Additionally, 13% of the men with P/LP germline mutations had low PSA ( < 4.0 ng/mL) at diagnosis. Our findings highlight the importance of the new guidelines and GGT importance in some men with PCa, particularly if they could benefit from a different therapeutic approach, or be influenced into cascade GGT. [Table: see text]

SAT-140 Determining the Undetermined: The Role of Tumor Tissue Staining for Interpretation of Inconclusive Genetic Testing Results in Patients with Pheochromocytomas and Paragangliomas

Background:Paragangliomas and pheochromocytomas are neuroendocrine tumors that can occur in several hereditary tumor syndromes. While these are generally rare, individuals with germline loss of function mutations in the succinate dehydrogenase (SDH) genes are at high risk of developing these tumours, with a penetrance of 70% by age 50. Functional SDHB acts as a tumor suppressor. Consequently, pathogenic mutations in the SDHB gene predispose to familial paraganglioma syndrome type 4, with high incidence of extra adrenal paragangliomas and high rates of metastasis. SDHB mutation carriers are also predisposed to developing tumors in other sites such as renal cell cancer, gastrointestinal stromal tumors and pituitary adenomas. Genetic testing for hereditary syndromes is recommended in patients who present with paragangliomas and pheochromocytomas, especially in those with aggressive tumours or who present at a young age. It is recommended that mutation carriers are monitored with routine clinical and imaging surveillance, and effort is made to identify and screen at-risk family members. In some cases, genetic testing can identify variants that are not clearly pathogenic or benign. In such “variants of undetermined significance”, immunohistochemistry or family history can be a helpful tool in discriminating between SDHB related and non-SDH-related pheochromocytomas and paragangliomas.Clinical case:We report on three families who presented with manifestations of paraganglioma syndrome and were found to have Variants of Uncertain Significance (VUSs) in the SDHB gene. Absence of SDHB staining was seen on tumour histopathology in two of the families; staining was not performed in the third. The proband in the third case initially presented at the age of 22 with a cardiac pheochromocytoma. Subsequently, her son was diagnosed with metastatic renal cancer at the age of 37. Genetic test results from both these patients identified a heterozygous VUS in SDHB. The son passed away from complications of his aggressive cancer shortly after diagnosis. Had familial screening and surveillance been initiated sooner in this family, this poor outcome may have been prevented.Conclusion:Our case highlights the important diagnostic dilemma that can arise in patients with VUSs in risk genes for hereditary pheochromocytomas and paragangliomas. While universally treating these VUSs as pathogenic would be costly, low-yield and potentially harmful, the incorporation of family history and tumour tissue staining for SDHB should be considered in all individuals with pheochromocytomas and paragangliomas to help guide interpretation of inconclusive genetic testing results, inform subsequent management and help predict risk for inheritance and recurrence.

Abstract P3-07-11: BRCA mutations and association with discordance in a large oncotype database

Abstract BACKGROUND: BRCA mutated tumors have been shown to be associated with higher recurrence scores as compared to BRCA negative breast cancer patients. Prior studies have demonstrated 7-19% discordance between oncotype RS and tumor grade (TG) in breast cancer patients. We developed a large oncotype database to determine if BRCA mutation status is associated with discordance. METHODS: We identified 723 patients (32 (4%) BRCA positive patients and 691 (96%) BRCA negative patients) with early-stage, hormone positive BC treated between 2006-2018, with tumor characteristics available for analysis. Discordance was defined as one or two-step difference between RS (low, intermediate, high risk) and TG (well (WD), moderately (MD) and poorly (PD) differentiated). BRCA status was defined as BRCA 1 deleterious mutation, BRCA 2 deleterious mutation, BRCA mutation of unknown type, BRCA variant of undetermined significance (VUS) or other mutation. N(%) or median were used to describe patients’ characteristics between groups and compared by Mann-Whitney U test at the confidence level of 5%. RESULTS: Among these patients, there were 32 patients (4% of total) who had any documented mutation or VUS. 16% had documented deleterious mutation in BRCA 1, 22% BRCA 2, 6% BRCA unknown type, 25% BRCA VUS, and 31% other VUS (most commonly CHEK2 and ATM). Median RS was 23.5 in patients with deleterious BRCA mutations (1, 2 or unknown) vs. 16 in patients in the BRCA negative database which was statistically significant (p<0.01). One step and two-step discordance was present in 46% and 8% respectively of patients with deleterious BRCA mutations vs. 53% and 11% respectively in the BRCA negative database. CONCLUSIONS: Patients with deleterious BRCA mutations demonstrated no difference in rates of discordance as compared to BRCA negative patients. We further demonstrated that patients with BRCA positive tumors display higher recurrence scores than patients with BRCA negative tumors. Citation Format: Julia Blanter, Brittney Zimmerman, Serena Tharakan, Krystal Cascetta, Meng Ru, Amy Tiersten. BRCA mutations and association with discordance in a large oncotype database [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-07-11.

Feasibility of circulating tumor DNA testing in hepatocellular carcinoma.

Advancement of technologies enabling clinical assessment of circulating tumor DNA (ctDNA) are allowing for assessment of tumor specific genetic alterations in patients. This holds incredible promise for early detection of disease, serial monitoring of tumor heterogeneity, elucidation of therapeutic targets, and evaluation of treatment response and mechanisms of resistance. Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is often diagnosed late, recurs commonly, and is often diagnosed based upon imaging features alone. A comprehensive evaluation of real-time evaluation of ctDNA in patients with HCC has thus far not been undertaken. From January 2015 to February 2018, 35 patients with biliary tract cancer (BTC) at the Mayo Clinic Comprehensive Cancer Center underwent ctDNA testing using a clinically available assay. The majority of samples were tested utilizing the 73-gene panel which includes somatic genomic targets, including complete or critical exon coverage in 30 and 40 genes, respectively; and in some, amplifications, fusions, and indels. A total of 44 samples were collected on these 35 patients, with >70% having stage 3 or 4 disease. Among all samples the median number of alterations per sample, excluding variants of undetermined significance (VUS), was 3.5, with a median allele frequency of 0.65%. A total of 122 unique genetic alterations, excluding VUS or synonymous alterations, were seen. The overall landscape of alterations is described. The top 10 genes altered in this cohort of patients, excluding VUS or synonymous alterations, were TP53 (18%), TERT (14%), CTNNB1 (13%), ARID1A (9%), MYC (5%), BRAF (4%), CCND1 (4%), CDK6 (4%), and MET (4%), and EGFR (3%). Herein, we describe feasibility of ctDNA testing and results from such testing in HCC patients undergoing ctDNA testing in a real-time clinical context. Patients with these cancers stand to benefit immensely from the use of ctDNA technologies, and concerted efforts at further investigation of such are critically needed.

Hormone receptor expression of colorectal cancer diagnosed during the peri-partum period.

BackgroundColorectal carcinoma (CRC) during the peri-partum period is challenging to diagnose due to the overlapping symptoms of CRC and pregnancy. This is the first case series to investigate clinicopathologic, hormonal and molecular features of CRC diagnosed during the peri-partum period. We hypothesized that advanced presentations of CRC could possibly be mitigated by pregnancy-related hormonal factors.MethodsWe conducted a retrospective review of five women diagnosed with CRC during the peri-partum period and studied the clinical and molecular features of their cancer.ResultsAll patients presented with stage IV CRC at diagnosis; three had primary tumors in the rectum and two had primary tumors in the sigmoid colon. The liver was the most common metastatic site (three of five women). Immunohistochemistry stains were negative for estrogen receptors alpha (ERα) and beta (ERβ), and one tumor demonstrated low-level positivity for PR (1%). Formalin-fixed and paraffin-embedded (FFPE) biopsies from each case were tested with next-generation sequencing and found that all tumors were mismatch repair (MMR) proficient, and three harbored a KRAS mutation. Germline testing showed no predisposition to CRC; however, several somatic variants of undetermined significance (VUS) were identified.DiscussionCRC in the peri-partum period poses significant risk factors for presentations with advanced disease due to diagnostic challenges. While our study provides no evidence that pathogenesis of CRC during pregnancy is driven by elevated estrogen and/or progesterone levels during pregnancy, additional putative etiologic factors, including placental growth factors, the immunosuppressive state of pregnancy and other physiologic processes during pregnancy, warrant future study.

Molecular comparison of primary colorectal cancer (pCRC) with peritoneal metastases (PM).

498 Background: PM from CRC are associated with poor outcomes; however, molecular differences are not defined. Methods: We compared tumor profiles of pCRC and PM patients (pts) from Caris Life Sciences. Testing included next-generation sequencing (NGS) of 592 genes, immunohistochemistry (IHC), copy number variants (CNV), microsatellite instability (MSI) and tumor mutational burden (TMB). Mutations were termed pathogenic (PATH) or variants of undetermined significance (VUS). TMB in mutations/Mb (MMB) was compared. Results: 617 pCRC and 348 PM pts had similar gender (55% male) and age (median 59). 232 pCRC were left-sided (LS), 189 right-sided (RS), 147 rectum (R) and 49 not otherwise specified (NOS); PM were 45 RS, 29 LS, 22 R and 252 NOS. For pts with IHC testing, expression was increased in PM in TOPO1 (62% v. 52%, p < 0.01), ERCC1 (27% v. 18%, p < 0.01) and MLH1 (96% v. 92%, p < 0.05) and decreased in PD-1 (36% v. 65%, p < 0.01), TOP2A (76% v. 100%, p < 0.01) and PTEN (64% v. 72%, p < 0.05). By sidedness, LS PM were more frequently TOP01 and PD-L1 and less commonly MGMT positive compared to pCRC. PTEN IHC was higher in R pCRC than PM. 7 CNVs were increased in PM ( ADGR2A2, CCND1, ELL, FGF3, FGF4, JAK3 and PDGFRB) and FLT3 CNVs were decreased. MYC CNVs were more common in RS PM compared to pCRC. No difference was seen in PM and pCRC PATHs in KRAS, BRAF, SMAD2, SMAD4, PTEN. PM had more PATHs in GNAS (8% v. 1%, p < 0.01) while pCRC PATHs were increased in APC (76% v. 48%, p < 0.01), TP53 (72% v. 53%, p < 0.01), ARID1A (29% v. 12%, p < 0.05), PIK3CA (22% v. 15%, p < 0.05) and FBXW7 (13% v. 7%, p < 0.01). LS PM had increased FLCN PATHs (12% v. 2%, p < 0.01); R PM had more PATHs in KMT2D (20% v. 1%, p < 0.01) and RNF43 (13% vs. 3%, p < 0.05). VUS were increased in 39/592 (7%) genes for PM compared to pCRC. No MSI or fusion difference was seen. 53% pCRC (median = 8) pts had TMB ≥8 MMB compared to 43% PMs (median = 7; p = 0.03); no TMB difference was seen for LS, RS or R subgroups. Conclusions: Compared to pCRC, PM had more PATHs in GNAS and less in classic CRC markers APC and TP53. While TMB was generally lower in PM, differences in IHC expression, CNV and VUSs may serve as biomarkers for PM requiring further study.

Abstract P2-09-06: Multi-gene panel testing for hereditary cancer risk

Abstract Background: Multi-gene panels are widely available for assessing hereditary cancer risk in high risk individuals. Due to the use of these panels, many genetic mutations other than BRCA 1 or 2 can be detected which can potentially affect management. This study presents the results of multi-gene panel testing performed at Beaumont Health System. Methods:All patients who underwent multi-gene panel testing at Beaumont Health System between November 1, 2012 and January 15, 2015 were included in this study. This cohort consisted of patients who met criteria for genetic testing due to personal or family history. All patients received comprehensive pre and post-test genetic counseling. The panels ranged from 5 to 43 genes associated with risk for breast and other cancers. Results: 653 multi-gene panel tests were performed. The majority of these consisted of either a 5 gene high risk breast panel (25%), an 18 gene moderate to high risk breast panel (21%), or a 9 gene high risk breast and gynecologic panel (17%). 184 variants of undetermined significance (VUS) were identified with a pooled VUS rate of 28%. Among the commonly used panels, there was a positive correlation between VUS rate and the number of genes included in the panel (r = 0.86, p = 0.01, Range 6% to 70%). A pathogenic mutation was identified in one or more genes in 65 (10%) panels for a total of 67 mutations. Of these, 17 mutations were in BRCA1 or BRCA2 gene. Fifty non-BRCA deleterious mutations were identified with the following frequencies: CHEK2(12), MUTYH(7 monoallelic, 1 biallelic), TP53(4), PTEN(4), ATM(4), MSH6(3), PALB2(3), MSH2(2), CDH1(2), APC(2), NF1(2), BARD1(2), MLH1(1) and PMS2(1). Of these non-BRCA mutations, 41(82%) had a significant impact on management. Conclusions: Our study demonstrates that multi-gene panel testing identifies several genes that can impact management and would likely not have been discovered by pedigree analysis alone. However, this added detection is associated with a higher VUS rate, especially using larger panels. Further research is needed to better define the role of multi-gene panel testing in high risk patients, with a focus on choosing appropriate genes, understanding the magnitude of cancer risk and delineating impact on management. Citation Format: Yadav S, Ladkany R, Fulbright J, Dreyfuss H, Reeves A, Campian S, Thomas V, Zakalik D. Multi-gene panel testing for hereditary cancer risk. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-06.